Isolation of human Dna2 endonuclease and characterization of its enzymatic properties Journal Article
| Authors: | Kim, J. H.; Kim, H. D.; Ryu, G. H.; Kim, D. H.; Hurwitz, J.; Seo, Y. S. |
| Article Title: | Isolation of human Dna2 endonuclease and characterization of its enzymatic properties |
| Abstract: | In eukaryotes, the creation of ligatable nicks in DNA from flap structures generated by DNA polymerase δ-catalyzed displacement DNA synthesis during Okazaki fragment processing depends on the combined action of Fen1 and Dna2. These two enzymes contain partially overlapping but distinct endonuclease activities. Dna2 is well-suited to process long flaps, which are converted to nicks by the subsequent action of Fen1. In this report, we purified human Dna2 as a recombinant protein from human cells transfected with the cDNA of the human homologue of Saccharomyces cerevisiae Dna2. We demonstrated that the purified human Dna2 enzyme contains intrinsic endonuclease and DNA-dependent ATPase activities, but is devoid of detectable DNA helicase activity. We determined a number of enzymatic properties of human Dna2 including its substrate specificity. When both 5′ and 3′ tailed ssDNAs were present in a substrate, such as a forked-structured one, both single-stranded regions were cleaved by human Dna2 (hDna2) with equal efficiency. Based on this and other properties of hDna2, it is likely that this enzyme facilitates the removal of 5′ and 3′ regions in equilibrating flaps that are likely to arise during the processing of Okazaki fragments in human cells. © 2006 Oxford University Press. |
| Keywords: | controlled study; human cell; promoter region; genetics; nonhuman; metabolism; cell line; enzyme activity; chemistry; dna; hybrid protein; genetic transfection; recombinant fusion proteins; enzyme analysis; saccharomyces cerevisiae; eukaryota; 5' untranslated region; nucleotide sequence; substrate specificity; recombinant protein; 3' untranslated region; helicase; okazaki fragments; flap endonuclease; single stranded dna; dna, single-stranded; flap endonucleases; structure analysis; enzyme specificity; adenosine triphosphatase; enzyme structure; isolation and purification; adenosine triphosphatases; enzyme purification; dna helicases; complementary dna; deoxyribonuclease; endodeoxyribonucleases; dna2 protein, human; enzyme isolation |
| Journal Title: | Nucleic Acids Research |
| Volume: | 34 |
| Issue: | 6 |
| ISSN: | 0305-1048 |
| Publisher: | Oxford University Press |
| Date Published: | 2006-01-01 |
| Start Page: | 1854 |
| End Page: | 1864 |
| Language: | English |
| DOI: | 10.1093/nar/gkl102 |
| PUBMED: | 16595799 |
| PROVIDER: | scopus |
| PMCID: | PMC1428795 |
| DOI/URL: | |
| Notes: | --- - "Cited By (since 1996): 26" - "Export Date: 4 June 2012" - "CODEN: NARHA" - "Source: Scopus" |
